Multiple molecular forms of invertase in maize smut infections

Physiological

Plant Pathology

(1980)

16,93-107

Multiple molecular forms of invertase in maize smut infections J. A. CALLOW, D. E. LONGS and E.D. LITHGOW Dept

of Plant Sciences, University

(Accepted

fw publication,

34

of L-seds, Leeds W2 9jT,

U.K.

1979)

Following infection of maize sudlings with the maize smut fungus, Ustilago maydis, a progressive increase in the absolute activity of acid invertase was detected over a 16 day period. Activity declined in uninfected seedlings over the same period. This increased activity in infected tissues was correlated with a low sucrose and high hexose content and, presumably, the enzyme serves to provide a supply of hexoses to the growing neoplastic tissues of the host, and to the fungal heterotroph. The major proportion of thii increased invertase activity was due to soluble, rather than cell wall-bound enzyme. A single soluble invertase component of mol. wt 100 000 was detected in uninfected tissues by gel electrophoresis and gel filtration. This component was also present in infected tissues but the major activity in these was due to an additional component of mol. wt 145 000. The latter was identical in mol. wt to one of two major invertase components present in cytoplasmic extracts of cultured haploid sporidia of U. maydis. A second major invertase of cultured U. myadis cells (of mol. wt 300 000) was not represented in infected maize tissues. It is suggested that the increased invertasc activity of smutted tissue is due to an enzyme of fungal origin, and to a lesser extent, a stimulation of host invertase. These conclusions differ markedly from those reported by other workers using the same host-parasite combination.

INTRODUCTION

It has frequently been observed that altered patterns of translocation in plants infected by biotrophic fungal pathogens result in the accumulation of host photosynthate, in particular, hexose sugars, starch or other polysaccharides, at the immediate sites of infection [18]. Sucrose is the major form in which carbon is tramlocated in higher plants, and in general there is a good correlation between a low level of sucrose in tissue and a high activity of acid invertase, the major enzyme responsible for the hydrolysis of sucrose to hexoses [2]. Investigations on three biotrophic infections [18] showed that in each case a substantial increase in acid invertase activity in infected tissues correlated with high levels of hexose sugars. It was therefore suggested that acid invertase is a key enzyme in the nutrition of biotrophic parasites inverting sucrose to the more readily absorbed hexoses and also providing substrates for the accumulation of insoluble polysaccharides in the host such as starch. The precise location of the increased invertase activity and its origins were not examined. Characterisation of enzymes in infected tissues ultimately requires that both components of the symbiosis be isolated and cultured separately. Physical separation of host and fungus is difficult in most cases of parasitism and many biotrophic tCurrent

address:

0048-4059/80/010093

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+ 15 $02.00/O

Foundation,

Lyttel Hall, NutfIeld, Rcdhill, Q 1980 Academic Press Inc.

Surrey. (London)

Limited

94

J. A. Callow,

D. E. Long and E. D. Lithgow

parasites cannot be grown with ease in axenic culture. For these reasons, invertase activity and carbohydrate metabolism in biotrophic infections of maize seedlings by the maize smut fungus, U&ago maydis, were examined. Ustilago species are readily cultured and can hydrolyse exogenous sucrose [II]. The infection of meristematic tissues of maize by Ustilago maydis results in tissue hyperplasia and gall formation [S, 7]. Microscopic studies revealed a transient accumulation of starch in mesophyll plastids [6]. Since these plastids do not normally store starch, it was suggested that infection induces changes in carbohydrate translocation patterns leading to temporary starch storage and potentially, therefore, implicating an active acid invertase. The present investigation was therefore undertaken to establish whether maize smut infections conform to the situation reported for other biotrophic infections where the activity of invertase increases, and to establish the extent to which host and fungal enzymes are involved. Whilst this work was in progress, Billett, Billett & Burnett, [4] also working with maize smut, reported an increased activity of acid invertase in neoplasms which they attributed to a host enzyme on the basis of electrophoresis. Using essentially the same materials and methods, we report in this paper a stimulation of invertase activity somewhat different in form to that previously reported. The major portion of this increased activity appears to be due to an enzyme of ‘fungal’ origin. MATERIALS AND METHODS Growth of plants and inoculation

Sporidial wild-type mating strains of Ustilago maydis (DC) Corda were grown to early stationary phase (lo8 cells cm -“) or mid-log phase, in shake culture at 30 “C [6]. Cultures of opposite mating type were then mixed and injected into the whorls of leaf sheath tissue of 5-day-old maize seedlings (,$a mays, L., hybrid corn cultivar SA4) as described previously [S]. Maize was grown in trays of proprietary soil-less compost (Bower’s Lincoln, U.K.) at 25°C with supplementary glasshouse illumination. Control plants were sham-inoculated with sterile distilled water. Extraction

and analysis of soluble carbohydrates

Samples of control and infected leaf sheath tissue were extracted into 80% ethanol and the soluble carbohydrates were analysed by g.1.c. of the trimethylsilyl derivatives [24] by methods previously described [17]. Determination

of total tissue invertase activity

Samples of transverse slices, 1 mm thick, taken from control and infected leaf sheath tissues at 2 day intervals after inoculation, were pre-treated with ice-cold ethyl acetate to rupture cell membranes, permitting exposure of exogenous substrate to the total cell invertase and removing endogenous substrates and products [3]. Pre-treated samples were then washed in ice-cold water before assaying for total invertase by incubation in sucrose medium, pH 5.5, as described previously [18]. The release of reducing sugar into the medium was assayed by the method of Nelson [ZO]. After incubation the tissue samples were washed and dried to constant weight.

Forms of invertase

in maize smut infections

95

Invertase activity was expressed as pg reducing sugar released mg -l dry wt h -l. All assays were conducted with triplicate sets of samples. Cellular distribution of invertase Control and infected leaf sheath tissues were extracted in a pestle and mortar with 0.2 M Tris-MES buffer pH 7.2 containing O-004 M 2-mercaptoethanol, at 4°C. The homogenate was centrifuged at 2000 g for 2 min and the crude ‘cell wall’ pellet retained. The supernatant fraction was washed three times with Tris-MES buffer, twice with 1 M NaCl to remove ionically-bound enzyme then twice with 0.1% Triton X-100 to solubilize membranes. The pellet was finally washed three times in Tris-MES buffer and retained as a ‘cell wall’ fraction. Aliquots of the particulate, cytoplasmic and cell wall fractions were incubated in 0.1 M sucrose in citratephosphate buffer pH 5.5 and assayed as above. Determination of soluble invertase Control and infected leaf sheath tissues were extracted in a Polytron blender with O-1 M phosphate buffer pH 7.2 containing 0.1 M NaCl and 0.004 M 2-mercaptoethanol. The homogenate was successively centrifuged at 5000 g for 15 min and 100 000 g for 60 min. The final supernatant fraction was diaiysed overnight against 0.05 M acetate buffer pH 5.0 and assayed for invertase activity in the same buffer as described above. Fungal invertase Sporidial cultures of haploid Ustilago maydis mating strains were grown on an orbital shaker at 30°C in the minimal medium described previously [6] but with sucrose replacing glucose. Cultures in log phase were used for the extraction of soluble cytoplasmic invertases. Cells were harvested by centrifugation at 10 000 g for 10 min, resuspended in 20 cm3 0.1 M Tris-MES buffer pH 7.4 containing O-004 M 2-mercaptoethanol then broken by three passages through an Aminco French Pressure Cell at 200 MPa. Unbroken cells and cell walls were removed by centrifugation at 10 000 g for 10 min and finer particulate material at 100 000 g for 60 min. The post-ribosomal 100 000 g supernatant was used as the cytoplasmic invertase preparation after dialysis against the appropriate buffer. Extracellular, secreted fungai invertase was prepared from older, 80 h cultures in stationary phase, by removing cells at 10 000 g for 10 min and dialysing the supernatant against 0.05 M citrate buffer pH 5.5. Electrophoretic separations of invertase Soluble extracts of control and infected maize tissues, and U&ago sporidia were layered on to 7.5 or 10% polyacrylamide gels in 20% (v/v) glycerol before electrophoresis in O-1 M Tris-glycine buffer pH 9.5 at 1 mA per gel for 10 min and 4 mA per gel for 3h. Gels were then frozen in solid COs and sliced into O-7 mm or 1 mm segments. Invertase activity in each segment was measured by incubating with O-5 cm3 0.1 M sucrose in 0.05 M citrate-phosphate or acetate buffer pH 5.0 to 5.5, at 30°C for 2 to 24 h. For periods longer than 6 h, one drop of chloroform was added to each tube as a bacteriostat. After removing the gel segments, reducing sugars

96

J. A. Callow, D. E. Long and E. D. Lithgow

released into the medium were assayed as before. incubated in the absence of sucrose as controls.

Replicate gels were sliced and

Sephadex G 150 geljiltration Soluble invertases were separated on columns of Sephadex G 150 (39 x 2.5 cm) in 0.1 M acetate buffer pH 5-O containing O-025 M NaCl with a flow-rate of 12 to 15 ems h -1. Fractions (4 cm*) were collected at 4°C. The columns were calibrated for molecular weight determinations using beef liver catalase (230 to 250 x lo3 daltons), rabbit muscle aldolase (140 to 150 x lo3 daltons), bovine serum albumin (65 to 70 x lo3 and 130 to 140 x lo3 daltons), ovalbumin (44 to 46 x 10s daltons) and cytochrome c (12.4 x lo3 daltons) [I]. All markers were purchased from Sigma Chemical Co. Enzyme activity in fractions was determined by incubating 0.375 ems of each fraction with 0.125 cm3 of O-8 M sucrose for various times at 30°C. Reactions were stopped with 0.5 cm3 Nelson’s copper reagent. Each sample was heated to boiling for 20 min, cooled, then O-5 ems arsenomolybdate reagent added [ZO]. Each sample was diluted as appropriate before reading at 520 nm. Control incubations for each fraction were performed in the absence of sucrose, or by stopping at time zero. @H optima of invertases Activities were determined in triplicate incubations over the pH range 3.6 to 7.5 in O-1 M buffer containing 0.025 M NaCl and O-2 M sucrose. Acetate buffer was used over the range 3.6 to 5.5, and phosphate buffer for the range 5.7 to 8-O. Control incubations for spontaneous sucrose hydrolysis and reducing sugar release from enzyme extract in the absence of sucrose, were carried out simultaneously with each assay and subtracted as appropriate. The sensitivity of the Nelson reducing sugar assay was found to be constant across the pH range employed with these buffers. RESULTS The time-course of events during maize smut infection followed that previously reported [S]. Within 4 days of inoculation, there was a marked inhibition of leaf expansion and widespread chlorosis. After 7 days sheathing leaf bases showed marked neoplasia, developing spore-containing galls within 14 days. The soluble carbohydrate composition of neoplastic leaf sheath tissues of infected maize was altered both quantitatively and qualitatively. On a dry wt basis, total soluble carbohydrate content of infected tissue, 10 days after inoculation, was 34 pg mg -1 compared with 13 pg mg -l in control tissue (unpublished observations). This is consistent with the changes in translocation patterns and accumulations of host photosynthetic products observed in other biotrophic infections [15, 16, 21, 221. This increased soluble carbohydrate content is partially accounted for by the accumulation of host hexoses, fructose and glucose, and fungal carbohydrates, trehalose, erythritol, arabitol and mannitol (Fig. 1). In comparison with control tissues the ratio of sucrose to hexoses was considerably decreased (from approximately O-52 to O-17 at day 10). Total invertase activity of uninfected tissues, as estimated by the ethyl acetate technique, declined over the period studied (Fig. 2). Only small differences in total

Forms of invertase

97

in maize smut infections IO 8

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6

0

4

8

12

16

20

24

28

Relention time (mid

Fm. 1. G.1.c. analyses of the TMS derivatives of soluble carbohydrates from (a) uninfected, (b) infected maize seedlings 10 days after infection. Equivalent tissue fresh weights were injected onto the column. Abbreviations: F, fructose; G, glucose (a and fl forms) ; &sucrose; El, erythritol; Al, arabitol; M, manmtol; T, trehalose.

FIG. 2. Activity of invertase determined by the ethyl acetate, wt basis, mean values of replicate

in uninfected (0) and infected (A) leaf sheath tissues, as total tissue technique. Activity expressed on a per unit dry determinations are marked with 95% confidence limits.

98

J. A. Callow, D. E. Long and E. D. Lithgow

invertase activity were detected in infected tissues until 4 days after infection when there was a marked, progressive increase in total activity so that by 12 days after infection, when leaf sheaths were grossly hypertrophic, activity in infected tissues had increased five-fold from the time of inoculation, and was 28 times greater than the activity in control tissues of the same age. Although criticised [4], in our hands the ethyl acetate, total tissue invertase technique has proved to be a reliable method for estimating trends in total invertase activity. A time-course analysis of soluble invertase showed very similar trends (Fig. 3) although detailed comparisons have not been made within the same experiment. c

36

% $ 32I,P

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x 2

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FIG. 3. Activity of soluble invertase extracted (A-A) leaf sheath tissues. Activity expressed per Valuol are the means of duplicate determinations.

TABLE

from unit

12

14

uninfected fresh weight

l

16 (@+) and infected of tissue extracted.

1

Cellukar distribution of acid inoertuse actioity in infected and uninfected leaf sheath tissue (enqme actioity expressed as pg reducing sugar mg-l dty wt h-l). Tku~ taken 8 akys afm itwc&ion (1Pdayold seedlings). Values conoerted fm orginal tissue dry wt using a fieshldry wt conversion ratio for each @be of tissue cell Uninfected sheath 8 days after inoculation Infected sheath 8 days after inoculation krude 2000 x g pellet, blOO 000 g pellet. c1 000 000 g supernatant.

walls”

Particulateb

13.8 ;;I.;%)

Solublec

Total

52.9

75.9 1168.5

(2011%) not salt and detergent-washed

at this stage.

Forms of invertase

in maize smut infections

99

The cellular location of the increased activity was investigated by fractionating tissue homogenates 8 days after infection. At this time, 70% of the total tissue invertase activity in controls was buffer-soluble, non-particulate (Table 1). Of the 15 to 18% retained by the cell wall fraction, approximately half could be rendered soluble with 1M NaCl and Triton X-100 (data not shown). The portion remaining firmly attached to cell walls represented only 10% of the total activity. The increased total invertase activity of infected tissues resulted from increases both in soluble and cell wall fractions (Table 1 and Fig. 3). Both cell wall and soluble activities increased 17-fold compared with uninfected tissues of the same age. Only 4% of the activity present in crude cell wall fractions of infected tissues could be removed by salt and detergent washes (data not shown). More detailed studies were carried out on the major, soluble fraction of invertase activity in control and infected tissues, and from U. maydis sporidia. In all cases pH optima were acidic, no alkaline invertases were detected (Fig. 4). Although Hatch

PH

Fro. 4. pH activity profiles of dialysed, soluble invertases (100 000 x g supernatants) isolated from uninfected maize (MC, l ), U. q&s sporidia (Fc, @), infected maize, 7 days after inoculation (Mi, A). Activity is expressed as a percentage of maximal activity at the optimal pH in 0.1 M acetate buffer (pH 3.6 to 5.5) or 0.1 M phosphate buffer (pH 5-7-8.0).

et al. [l.?] reported that tris has an inhibitory effect on sugar cane invertases, in particular alkaline or neutral invertases, it is unlikely that our failure to detect alkaline invertases is due to Tris since all extracts, including those made in Tris-based buffers, were extensively dialysed against water or acetate buffer, before assay, or in later experiments, before gel filtration. In general form, the pH profile of the total soluble invertase of infected maize was intermediate between those of the uninfected maize and fungal extracts. Uninfected maize invertase has a well-defined optimum at pH 4.5 to 5-O with markedly reduced activity at higher pH. In contrast, although total soluble invertase of infected maize had an optimal activity very similar to that of uninfected maize, considerable activity remained at higher pH values, as is also the case for the enzyme from Ustilago sporidia. This suggests that total invertase

100

J. A. Callow, D. E. Long and E. D. Lithgow

of infected maize has contributions both from host and parasite although it must be emphasised that the activity profiles were based on crude, total soluble invertase samples and cannot be considered to be diagnostic without other data. Following gel electrophoresis on 10% polyacrylamide at pH 9.5, cytoplasmic extracts of uninfected plants contained a single acid invertase component of low mobility [Fig. 5(a)]. I n contrast, extracts of infected tissues produced two peaks of activity, one corresponding to that of uninfected plant extracts but with enhanced activity (22 I.tg reducing sugar equivalents g -l fresh wt h -l in control tissue, 63 ug g -l fresh wt h -l in infected tissue), and a second broad band of higher activity with a greater electrophoretic mobility [Fig. 5(b)]. The soluble, cytoplasmic invertase preparation of U. maydis and samples of culture

lb)

4

I

2 Gel length

3

(cm)

FIG. 5. Gel electrophoresis of soluble invertase preparations from, (a) leaf sheath tissues of uninfected maize seedlings, 10 days after inoculation with sterile water (MC, dialysed 100 000 g supernatant) ; (b) leaf sheath tissues of infected seedlings, 10 days after inoculation with U. mq& (Mi, dialyscd 100 000 g supernatant); (c) culture supernatant of U. maydis sporidia, early stationary phase (Fs, dialysed 10 000 g supernatant). Gels were 10% polyacrylamide at pH 9.5, run for 3 h at 4 mA per tube. On completion of electrophoresis, gels were frozen, sliced into 0.7 mm segments and invertase activity in each segment determined. Results are corrected for release of reducing sugar in the absence of sucrose, as determined from replicate gels.

Forms of invertase

in maize smut infections

101

filtrate were also subjected to electrophoresis under the same conditions as the plant extracts. Both fimgal enzyme preparations produced a single band of activity on gels, with mobilities similar but not identical to the major, fast-running component present on gels of infected plant extracts [Figs 5(c) and 71. This difference was observed consistently in a number of separations, and was confirmed by co-electrophoresis of the major invertase of infected maize and the extracellular, secreted invertase (Fig. 6). Since the two preparations were adjusted to contain approximately

Gel length

km)

FIG. 6. Electrophoresisofinvertasepreparationsfrominfected maizeleafsheathtissue (oM~, 10 days after inoculation, diaIysed 100 000 x g supernatant), and culture supematants of U. may&s sporidia (0 Fs, early stationary phase, dialysed 10 000 g supematant). In (a), both samples were run separately, sliced and activity of invertase determined. The two sets of results are plotted on the same graph. In (b), the gel segments containing the U. maydis supernatant invertase from a replicate gel, were placed on top of a fresh gel and co-electrophoresed with infected plant extract. The gel was then frozen, sliced and invertase distribution determined. All gels were 10% polyacrylamide, pH 9.5, run for 3 h at 4 mA per tube.

equal activities, the minor, low mobility component of the infected plant extract was not detectable. Thus it was possible to confirm that the major enzyme of infected tissue was distinct from both the secreted fungal invertase and the cytoplasmic fungal enzyme since the last two enzymes had identical electrophoretic mobilities (Figs 6 and 7). A comparison of the electrophoretic mobiities of the cytoplasmic invertases of the two mating strains of Ustilago maydis used to infect the maize seedlings, revealed no differences. Further separations were carried out by Sephadex G 150 gel filtration. Again, the soluble invertase of uninfected maize leaf sheath tissues fractionated as a single component of molecular weight 100 000 daltons [Fig. 8(c)]. In contrast, infected

J. A. Callow,

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D. E. Long and E. D. Lithgow

maize extracts showed three peaks of activity [Fig. 8(d)]. The major peak (Mi,) of mol. wt 145 000 daltons accounted for approximately 60% of the total activity. A second, smaller peak (Mi,) had a molecular weight of 100 000, i.e. identical to the control enzyme peak, and a third peak, present in only very low activity (Mis) had a mol. wt of 45 000 to 50 000 daltons. The soluble, cytoplasmic fraction of U. muydis sporidia also showed three peaks of activity [Fig. 8(a)], two of which (Fcr and Fc,) were present as major activities. Fcs had the same molecular weight as Mi, and the third, minor component, Fc, ,had the same molecular weight as Mi,. The major Fc, component was, however, unique to the cultured fungus, not being represented in extracts of infected plants and with a high mol. wt of 300 000 daltons. Finally, in the supernatant from cultured U. maydis the main activity had a peak identical to Fc, in molecular weight, with a broad spread of activity at lower molecular weights covering the region equivalent to Fc, [Fig. 8(b)]. Activity in culture supernatants was low except in older cultures in stationary or senescent phases.

I

2

3

4

Gel length (cm1

FIG. 7. Gel electrophoresis of soluble invertase preparations from (a) leaf sheath tissues of infected plants, 10 days after inoculation (Mi, dialysed 100 000 g supernatant), (b) cytoplasmic extract of U. mo$~ sporidia in log phase (Fc, dia.lysed 100 000 g supernatant). Gels were 10% polyacrylamide, pH 9-5, run for 3 h at 4 mA per tube. After electrophoresis gels were frozen, sliced into 1 mm segments and invertase distribution determined. Reducing sugar was not released from control, replicate gels incubated in the absence of sucrose.

Forms of invertase in maize smut infections

103

l-2

0.8

0.4

0 0.03 0.02 0.01 0 p

I-0

I,.\

0 W

!

20

30 Fraction

40

no.

FIG. 8. Sephadex G 150 gel filtration of cytoplasmic extract of (a) U. may& sporidia (dialysed, 100 000 g supematant); (b) dialysed, culture supematant of U. mydis sporidia; (c) dialysed 100 000 g supernatant of soluble extract of leaf sheath tissue of uninfected maize seedlings; (d) dialysed 100 000 g supernatant of cytoplasmic extract of infected leaf sheath tissues of maize, 10 days after inoculation. Each sample was separated on the same column (39 x 2-5 cm) in 0.1 M acetate buffer, pH 5.0, containing O-025 M NaCl, with a flow-rate of 12 to 15 cm* h-1, collecting 4 cm* fkactions at 4°C. Aliquots of each fraction were used for invertase assays. Activities are presented as A Eon0 a&r subtraction of zero time and minus sucrose controls. The cohmm void volume is indicated (Vo) .

104

J. A. Callow, D. E. Long and E. D. Lithgow

DISCUSSION Substantial changes in the carbohydrate content of plant tissues infected by biotrophic fungal parasites are indicative of alterations in the normal translocatory patterns of the host. Although detailed quantitative analyses were not made in this study, it is clear from the substantial growth in size of the infected tissue and the accumulation of starch and other carbohydrates, that photosynthetic products are accumulated in the neoplastic leaf sheath tissues of infected plants, within which the fungus is localised [q. More detailed studies by others [5l confirm this and, in other smut infections such as that caused by U&ago nuda in wheat [9, lo], similar changes in carbohydrate distribution were noted, although they were not accompanied by marked tissue hyperplasia. In maize smut infections, assimilates directed to sites of infection, presumably in the form of sucrose, are utilized both to support fungal growth and proliferation of host tissue. The g.1.c. analyses of soluble carbohydrates are consistent with these two growth components. On the one hand, the presence in infected tissue of the typical fungal metabolites, trehalose, erythritol, arabitol and mannitol, and their absence from uninfected tissue is in accordance with the general thesis that, in many forms of symbiosis, formation of gradients through the conversion of host assimilates to metabolites unique to the heterotrophic partner, aids the continued flow of carbon compounds to the heterotroph [Z?]. On the other hand, the lower sucrose: hexose ratio in the infected tissue is characteristic of tissues in which there is a high demand for hexoses, such as those which are growing rapidly [Z] or those which are infected by biotrophic fungal parasites [18]. On a priori grounds then, it might be anticipated that invertase is an important enzyme in maize smut disease and the marked changes in activity reported here suggests that this is so. On a dry wt basis, after an initial lag, there was a substantial increase in activity over a 12 day period, both in absolute activity and in terms of activity relative to uninfected seedlings. Since both host and fungal components of the maize smut infection are potentially utilizing invertase to supply hexoses for growth, an examination of the source of the increased activity is particularly pertinent. In the present investigation the major fraction of the invertase activity in infected tissues was shown by gel electrophoresis and gel filtration to be due to an enzyme of quite different properties to the single invertase component present in uninfected tissue. Indeed, in terms of molecular weight, the major enzyme of infected tissue (Mi,) was identical to one of the two major invertase components present in U. maydis sporidia (FcJ. The minor invertase component of smutted tissues (Mi,) was identical in fractionation properties to the single enzyme component of uninfected tissue and it is clear that a small part of the overall increase in total invertase activity in infected tissues can be ascribed to this host enzyme. In yeast and Neurospora [13, 191 invertase occurs both within the fungal protoplast and outside, either in the wall itself or in the paramural space. A portion of the cellular invertase is thus located in direct contact with the external environment where it serves to initiate the metabolism of sucrose by splitting the disaccharide into its constituent monosaccharides before their uptake [S, 231. In yeast, the dual location of invertase is related to the presence of two molecular forms of the enzyme, an external high mol. wt mannan-protein and an internal low mol. wt protein

Forms of invertase

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105

without mannan [la]. The apoprotein portion of the external glycoprotein is similar to, but not precisely identical with, the internal protein. In .Neurosporacrassa again two forms of invertase are present but, here, the ‘heavy’ enzyme appears to be a polymer of enzymically-active ‘light’ sub-units with a reversible transition occurring between the two forms [19]. This transition appears to be quite slow under physiological conditions. In cultured haploid sporidia of U. maydis, the precise relationship between the major invertases present is not clear. Since one is twice the molecular weight of the other a monomer:dimer equilibrium similar to that in Neurospora may be operative, but preliminary attempts to alter this equilibrium have not been successful. If, as suggested above, the major invertase of infected tissue corresponds to Fc,, the low molecular weight invertase of cultured 17. maydis, then an important question concerns the factors which control the presence and distribution of the two forms of U. maydis invertase at different morphological and life-history stages. l-7.maydis in its infective, dikaryotic state has a different morphology to cultured, haploid sporidia and exists in a different environment, factors which may result in differential enzyme synthesis or affect the transition between two forms of the enzyme in equilibrium. Alternatively, it may be that in the pathological state, the fungus actively secretes invertase into the host tissues with differential secretion in favour of the low molecular weight form. In Neurospora different cell wall properties are known to affect the proportion of ‘heavy’ and ‘light’ enzymes secreted [25]. The results and conclusions of this paper differ markedly from those reported by others studying the same disease in a number of respects. Billett et al. [4] reported increased activities of the major soluble invertase fraction in infected tissues relative to the activity in control tissues. However, this increase was based solely on decreasing control activities, the absolute enzyme activity in infected tissues showed no increasing overall trend with time. An early increase in activity in infected maize one day after inoculation and before symptom expression, claimed by these authors, is difficult to substantiate since these authors did not present zero-time data. In addition, marked qualitative differences are apparent. Billett et al. [4], on the basis of in situ localization of invertases on polyacrylamide gels only, demonstrated that infected tissues contained a single invertase component with identical mobility to one of two invertase bands detected in extracts of uninfected plants, and quite different from any enzyme produced by cultured U. maydis cells. Furthermore, from culture supernatants and soluble sporidial extracts of U. maydis these authors were able to detect two quite discrete forms of fungal invertase, one from cytoplasmic extracts with a low electrophoretic mobility, the other from culture supernatants with a greater mobility. In the present paper we have described the separation of two major invertases from sporidial extracts of the fungus and it has not proved possible to distinguish any preferential secretion of either of these two enzymes into the culture supernatant. Indeed, in the present study, extracellular activity in supernatants from cultures grown on sucrose as the sole carbon source was very low, except in senescent cultures. Thus, the significance of this particular form of invertase in the nutrition of the fungus is not clear. The lack of clear resolution of invertases in this culture supernatant fraction on Sephadex Gl50 may suggest that the source

J. A. Callow,

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D. E. Long and E. D. Lithgow

of this supernatant activity is turnover of wall-bound enzyme with a certain amount of proteolysis occurring. The basis of the different results in these two reports is not known. It is unlikely that varietal and strain differences would produce such radically different results and conclusions. There are, however, differences in the nature, and possibly, intensity of infection between the two reports. Billett et al. [4] infected lo-day-old maize seedlings and analysed the basal areas of the fourth leaf, whereas in the present study, infection of 5-day-old seedlings, with all leaves still tightly rolled, essentially restricts lamina development and produces extensive neoplasia of the leaf sheath tissue [6]. The latter tissue was examined in the present study. However, whist it is possible that these differences would have a marked effect on the quantitative results, it is perhaps less likely that they would so radically affect the qualitative aspects. Billett et al. [4l concluded that increased invertase activity in smutted maize was due to a stimulation of an enzyme of host origin. Whilst the present study would not preclude the appearance of a novel host enzyme in infected tissue, the fractionation results strongly suggest that the major portion of the increased activity in our experiments is due to an enzyme of fimgal origin. Definitive proof of this rests upon more complete characterization of the enzymes and the application of serological techniques. Portions of this investigation

were supported by grants from the ARC and SRC.

REFERENCES 1. ANDREWS, P. (1965). The gel filtration behaviour of proteins related to their molecular weights over a wide range. Biochemical 30uwm196,585-606. 2. AP REES, T. (1974). Pathways of carbohydrate breakdown in higher plants. In MTP Znternutional Review of Science, Biochemistry Series I, Vol. 11, Plant Biochemistty, Ed. by D. H. Northcote, Butterworths, London. 3. BACON, J. S. D., MACDONALD, I. R. & KNIGHT, A. H. (1965). The development of invertase activity in slices of the root of Beta vulgaris, L., washed under aseptic conditions. Biochemical Journal 34, 175-182. 4. BILLETT, E. E., BILLEIT, M. A. & BIJRNE~, J. H. [ 1977). Stimulation of maize invertase activity following infection by U&ago maydis. Phytochemistp 16, 1163-I 160. 5. BILLET, E. E. & BURNETT, J. H. (1978). The host-parasite physiology of the maize smut fungus, Ustikrgo maydis II. Translocation of 14C-labelled assimilates in smutted maize plants. Physiological Plant Pathology 12, 103-I 12. 6. CALLOW, J. A. & LING, I. T. (1973). Histology of neoplasms and chlorotic lesions in maize seedlings following injection of sporidia of Ustilago mcydis (DC), Corda. Physiological Plant Pathology 3, 489-494. 7. ckmISTENsEN, J. J. (1963). Corn smut caused by Ustilugo maydis. American Phytopathological Society Monograph No. 2. 8. DE LA Fuzrrrz, G. & SOLS, A. C. ( 1962). Transport of sugars in yeasts, II. mechanisms of utilisation of disaccharides and related glyccnides. Biochimica et Biophysics Acta 56,49-62. 9. GAUNT, R. E. & MANNERS,J. G. (1971). Host-parasite relations in loose smut of wheat, II. The distribution of W-labelled assimilates. Annals of Botany, NS. 35, 1141-I 150. 10. GAUNT, R. E. & MANNERS, J. G. (1971). Host-parasite relations in loose smut ofwheat. III. The utilization of r”C-labelled assimilates. Annals of Botany, .N.X 35, 1151-1161. 11. GAUNT, R. E. & MANNERS, J. G. (1973). The production of trehalose and polyols by Ustilugo nudu in culture, and their utilization in healthy and infected wheat plants. flew Phytologist 72,

32 l-327. 12.

M. I. Studies

HATCH,

D., SAanzn, on enzymes

J. A. & GLASZIOU, K. T. (1963). Sugar of the cycle. Plafit Physiology 38,338-343.

accumulation

in sugar

cane.

Forms of invertase

in maize smut infections

107

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